Hepatitis E virus ORF3 is a functional ion channel required for release of infectious particles

Abstract

<p id="d1918768e209">Hepatitis E virus (HEV) is responsible for an estimated 20 million enterically transmitted cases of viral hepatitis globally. Here, we demonstrate that one of HEV’s three major gene products, ORF3, is an ion channel. Deletion of ORF3 abrogates release of infectious virions, and we show that viral egress can be rescued by expressing the influenza A virus (IAV) matrix-2 protein <i>in trans</i>. Expression of ORF3 facilitates ion flux across the plasma membrane, providing direct evidence for its viroporin activity. We identify regions within ORF3 abrogating both ion channel activity and particle release, thereby linking these two processes for a quasienveloped human virus and providing an attractive potential target for antiviral drug development. </p><p class="first" id="d1918768e215">Hepatitis E virus (HEV) is the leading cause of enterically transmitted viral hepatitis globally. Of HEV’s three ORFs, the function of ORF3 has remained elusive. Here, we demonstrate that via homophilic interactions ORF3 forms multimeric complexes associated with intracellular endoplasmic reticulum (ER)-derived membranes. HEV ORF3 shares several structural features with class I viroporins, and the function of HEV ORF3 can be maintained by replacing it with the well-characterized viroporin influenza A virus (IAV) matrix-2 protein. ORF3’s ion channel function is further evidenced by its ability to mediate ionic currents when expressed in <i>Xenopus laevis</i> oocytes. Furthermore, we identified several positions in ORF3 critical for its formation of multimeric complexes, ion channel activity, and, ultimately, release of infectious particles. Collectively, our data demonstrate a previously undescribed function of HEV ORF3 as a viroporin, which may serve as an attractive target in developing direct-acting antivirals. </p>